There is a fundamental gap in understanding how Vibrio cholerae is able to rapidly adapt to and multiply within both aquatic environments and the host gastrointestinal tract. Understanding the entire life cycle of V. cholerae, specifically how it modulates its physiology to adjust to differet environments, is critical to enhancing combat against this pathogen. The long-term goal of this research is to understand how V. cholerae adapts to different carbon sources and thereby persists in varying niches. The objective of this application is to identify and characterize genetc mechanisms by which V. cholerae synchronizes its physiology with available carbon sources. The central hypothesis of this proposal is that V. cholerae relies on a suite of small regulatory RNAs (sRNAs) to adjust to changing environmental conditions. There is growing evidence that in many bacteria, including V. cholerae, sRNA regulatory circuits modulate metabolic and behavioral processes. Thus, to truly understand how V. cholerae is able to adjust its physiology for long-term persistence, the multiple sRNAs involved must be identified and characterized. This project will address this need through three specific aims: 1) Determine how expression of the sRNA MtlS is regulated; 2) Characterize the mechanism through which the mannitol permease, MtlA, is degraded when mannitol is no longer present; and 3) Determine the in vivo function of the sRNA IGR4. Under the first aim, the regulation of an sRNA involved in mannitol metabolism, MtlS, will be studied using a transcriptional fusion between the mtlS promoter region and lacZ; genetic screens will lead to the identification and characterization of regulators of mtlS expression. Under the second aim, mass spectrometry-based quantitative proteomics will be used to identify proteins that are up-regulated upon expression of MtlS. These proteins will then be characterized with regard to their effect on the stability of the mannitol transporter MtlA. Under the third aim, biochemical assays will be used to test the working hypothesis that the sRNA IGR4 down-regulates synthesis of the Cra protein, a repressor of many glycolytic genes, including mtlA. Preliminary data support the working hypotheses in this application and establish that the proposed experiments are feasible in the applicant's hands. The proposed research is significant because it will identify components of carbon acquisition and metabolism that may inform the development of new therapeutics or vaccines that are more effective than those currently available. Indeed, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) - the primary transporter of many carbohydrates in bacteria and the major system that will be studied in this proposal - is absent from eukaryotes and has been described as an ideal target for antimicrobial agents. The approach is innovative because it seeks to investigate the persistence of V. cholerae by defining new regulatory circuits that control the PTS in this pathogen.